Cryopreservation is the most common approach for long term cell storage. The critical step for cryopreservation is the addition of the cryoprotective agents, such as dimethyl sulphoxide (DMSO) or glycerol and most of them toxic for the thawed cells. Most slow-cooling protocols accept as gold standard the use of 5 to 15% of DMSO for cryoprotectant agent and a controlled freezing temperature at a cooling rate of 1° C. per minute in order to avoid ice crystals formation that would damage the cells. Another alternative to minimize cryodamage is to avoid ice crystallization through the use of vitrification protocols, wherein the temperature is rapidly decreased to vapour or liquid phase of liquid nitrogen.
Cryoprotective agents do protect by modifying the freezing behavior of cells, specifically affecting the rates of water transport and ice crystal formation across the cell membrane.
For successful cryopreservation of most cell types a homogeneous cooling rate through the whole sample is crucial. Large volumes of cryopreservation solutions are subjected to temperature gradients that lead to non-homogeneous cooling rates. Routine procedures usually involve slow cooling, typically at a controlled freezing rate of 1 to 2° C./min. This is always followed by rapid thawing.
To freeze a large volume of cells, the dimensions of the package are also important. Ice crystallization is a strongly exothermic event, so freezing a large volume of an aqueous cell suspension involves the removal of a large quantity of heat energy. The rate at which heat is removed from the cell suspension as it is frozen will therefore be a large factor in determining how rapidly ice can form. If heat removal is inefficient, the temperature in the center of volume may reach a plateau close to the melting temperature of ice in the solution. For a large package, this would cause a substantial differentiation in cell environment across the sample during freezing, as the freezing profile at the surface would be quite different from that at the center (Avis K E & Wagner C M, 1999, Cryopreservation Applications in Pharmaceuticals and Biotechnology. Drug Manufacturing Technology Series, Volume 5, p-181-313: Interpharm Press).
U.S. Pat. No. 5,863,715 A tries to tackle this problem using a flexible container, a freezer bag holder that maintains the cross-sectional area small enough to allow a uniform heat transfer throughout all regions of the said bag. Drawbacks to this method include the need for a large storage space for such amount of cells, implying extra costs. Additionally, cryobags may also break off during handling and storage at very low temperatures.
US 2006019233 teaches how to reduce the freezing solution volume by concentrating at densities between 3×107 and 5×108 blood cells per ml, increasing in turn the rate of DMSO. Drawbacks of the method are thus related with the toxic characteristics of the cryogenic agent rendering cell viability limitations ranging from 60 to 90%, and also cell density limitations for particular cell types such as non-blood related cells. This method teaches away from the present invention, as the latter removes the excess of cryogenic agent in order to diminish its rate per cell in the freezing vial.
Some cell types are more sensitive to cryopreservation, showing very poor survival rates. Ware reported that increasing the liquid volume in the vial from 0.25 ml to 1 ml reduces cell survival to roughly 25% (Ware et al, “Controlled-rate freezing of human ES cells”. Biotechniques 38, 879-883 June 2005). Ware concluded that loss of efficiency is associated to the slower thaw rates of large-volume containers than those of smaller volumes container, i.e. vials versus straws. A rapid thaw was found to be critical for a successful cryopreservation.
Some basic thawing conditions are established in the art as current standard protocols for the best recovery of the cells. These protocols include temperature optimization and dilution of the cryoprotectant solution right after thawing by adding fresh cell culture medium for the recovery of the cells in an appropriate osmotic environment. As an alternative, the cryoprotective agent can be removed. The reconstitution steps involved in these procedures can provoke cell damage due to swelling when eluting the cryoprotective agent. In order to solve this problem, the present invention is able to keep an optimal cryoprotectant percentage and cell concentration ratio in the freezing solution during the equilibration time. The cryoprotectant agent enters the cells in an amount enough to be effective, the excess thereof is however left out in the medium and removed just before freezing. Later, the progressive dilution at thawing will greatly soften the cellular osmotic shock.
In order to avoid exposure of the cells and to preserve sterility, several proposals are described in the art. These comprise devices such as Ensura-Sep™ that uses a single centrifugation step using proprietary canisters, or more sophisticated and expensive solutions such as Cytomate cell processing system from Baxter (Calmels et al, “Preclinical evaluation of an automated closed fluid management device: Cytomate, for washing out DMSO from hematopoietic stem cell grafts after thawing” Bone Marrow Transplant. 2003 May; 31(9):823-8.) or the Sepax® system (Rodriguez L. et al, “Washing of cord blood grafts after thawing: high cell recovery using an automated and closed system.” Vox Sang 2004 October; 87(3):165-72) wherein cells are transferred from bag to bag in enclosed systems.
EP 2471359 A1 teaches a method that avoids the need of incorporating new fresh medium to the thawed cells for the dilution of the cryoprotective agent in high throughput microplate vessels. This is achieved by the previous freezing of an extra layer of culture medium in addition to the frozen cell solution. Although this method offers a lack of manipulation and increases cell viability after thawing due to the minimization of the osmotic shock, the amount of cells to be frozen remains restricted.
The problem of the art is then to provide a cryopreservation method of higher cell viability rates after thawing, suitable for large amount of cells. The solution provided by the present invention is to perform a concentration step after or during the equilibration of the cells with the cryoprotectant agent, minimizing the rate of the latter in the freezing solution, which increases viability in a surprising degree. Further, the freezing of these concentrated cells in a high surface to volume ratio allows a more homogeneous cooling and thawing rate that will optimize viability rates.